Methods and compositions for the specific inhibition of transthyretin (TTR) by double stranded RNA

Abstract

This invention relates to compounds, compositions, and methods useful for reducing transth:yretin (TTR) target RNA and protein levels via use of dsRNAs, e.g., Dicer substrate siRNA (DsiRNA) agents.

We claim: 
     
       1. A double stranded nucleic acid (dsNA) comprising first and second oligonucleotide strands, wherein the first strand is 15-66 nucleotides in length and the second strand is 20-66 nucleotides in length, wherein the second oligonucleotide strand has a region of complementary to a target transthyretin mRNA sequence selected from SEQ ID NOs: 2052, 2054, 2060, 2062, 2064, 2066, 2068, 2084, 2085, and 2087 and is capable of reducing expression of transthyretin mRNA containing the target mRNA sequence when the dsNA is introduced into a mammalian cell, wherein the second oligonucleotide strand comprises a sequence selected from the group consisting of SEQ ID NOs: 516, 518, 524, 526, 528, 530, 532, 548, 549 and 551, and wherein the dsNA comprises RNA and has at least one modified nucleotide. 
     
     
       2. The dsNA of  claim 1 , wherein the second oligonucleotide strand forms a duplex region of 19-21 base pairs or 21-25 base pairs in length with the first oligonucleotide strand. 
     
     
       3. The dsNA of  claim 1 , wherein the second oligonucleotide strand forms a duplex region of at least 25 base pairs in length with the first oligonucleotide strand. 
     
     
       4. The dsNA of  claim 2 , wherein the second oligonucleotide strand has a 3′-overhang of 2 nucleotides when the second oligonucleotide forms a duplex region with the first oligonucleotide strand. 
     
     
       5. The dsNA of  claim 1 , wherein the 3′ end of the first oligonucleotide strand and the 5′ end of the second oligonucleotide strand are joined by a polynucleotide sequence comprising ribonucleotides, deoxyribonucleotides or both, optionally wherein the polynucleotide sequence comprises a tetraloop sequence. 
     
     
       6. The dsNA of  claim 1 , wherein the modified nucleotide is selected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH 2 —O-2′-bridge, 4′-(CH 2 ) 2 —O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methlycarbamate). 
     
     
       7. The dsNA of  claim 1 , wherein the modified nucleotide is selected from the group consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3′-deoxyadenosine (cordycepin), a 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxyinosine (ddI), a 2′,3′-dideoxy-3′-thiacytidine (3TC), a 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotide of 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxy-3′-thiacytidine (3TC) and a monophosphate nucleotide of 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkyl ribonucleotide, a 2′-O-methyl ribonucleotide, a 2′-amino ribonucleotide, a 2′-fluoro ribonucleotide, and a locked nucleic acid. 
     
     
       8. The dsNA of  claim 1 , comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
       9. The dsNA of  claim 1 , wherein the dsNA is attached to a targeting ligand selected from the group consisting of a GalNAc moiety, a cholesterol, and a cholesterol targeting ligand. 
     
     
       10. The dsNA of  claim 1 , wherein the first oligonucleotide strand comprises a sequence selected from the group consisting of SEQ ID Nos: 132, 134, 140, 142, 144, 146, 148, 164, 165, and 167. 
     
     
       11. The dsNA of  claim 1 , wherein the first and second oligonucleotides comprise a pair of modified oligonucleotides defined by a TTR # and corresponding sequences selected from TTR-422, SEQ ID NO: 132 and SEQ ID NO:516; TTR-424, SEQ ID NO: 134 and SEQ ID NO: 518; TTR-430, SEQ ID NO: 140 and SEQ ID NO: 524: TTR-432, SEQ ID NO: 142 and SEQ ID NO: 526; TTR-434, SEQ ID NO: 144 and SEQ ID NO: 528; TTR-436, SEQ ID NO: 146 and SEQ ID NO: 530; TTR-439, SEQ ID NO: 148 and SEQ ID NO: 532; TTR-483, SEQ ID NO: 164 and SEQ ID NO: 548; TTR-484, SEQ ID NO: 165 and SEQ ID NO: 549; and TTR-489, SEQ ID NO: 1667 and SEQ ID NO: 551. 
     
     
       12. A pharmaceutical composition comprising the dsNA of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
       13. A method for reducing expression of a target transthyretin mRNA in a mammal, comprising administering a dsNA of  claim 1  to a mammal in an amount sufficient to reduce expression of a target transthyretin mRNA in the mammal. 
     
     
       14. A method for treating or preventing a transthyretin-associated disease or disorder in a patient, comprising administering to the patient in need thereof an amount of a dsNA of  claim 1  effective to treat or prevent the disease or disorder in the patient. 
     
     
       15. The method of  claim 14 , wherein the disease or disorder is amyloidosis.